Maria Elena Pero

Productive performance and blood profiles of laying hens fed Hermetia illucens larvae meal as total replacement of soybean meal from 24 to 45 weeks of age

&NA; The aim of the research was to study the effects of an insect meal from Hermetia illucens larvae (HILM) as complete replacement of soybean meal (SBM) on productive performance and blood profiles of laying hens, from 24 to 45 wk of age. A total of 108 24‐week‐old Lohmann Brown Classic laying hens was equally divided into 2 groups (54 hens/group, 9 replicates of 6 hens/group). From 24 to 45 wk of age, the groups were fed 2 different isoproteic and isoenergetic diets: the control group (SBM) was fed a corn‐soybean meal based diet, while in the HILM group the soybean meal was completely replaced by Hermetia illucens larvae meal. Feed intake, number of eggs produced, and egg weight were recorded weekly along the trial. At 45 wk of age, blood samples were collected from 2 hens per replicate. The use of HIML led to a more favorable (P < 0.01) feed conversion ratio in hens but lay percentage, feed intake, average egg weight, and egg mass were higher (P < 0.01) in hens fed the SBM diet. Hens fed insect meal produced a higher percentage of eggs from small (S), medium (M), and extra‐large (XL) classes (P < 0.01) than SBM, while the SBM group had a higher percentage of eggs from the large (L) class (P < 0.01). The levels of globulin and albumin to globulin ratio were, respectively, higher and lower (P < 0.05) in HILM than the SBM group. Cholesterol and triglycerides were higher (P < 0.05 and P < 0.01, respectively) in hens from SBM than in the HILM group. Blood levels of Ca were higher (P < 0.01) in hens fed insect meal, while creatinine was higher (P < 0.01) in blood of hens fed SBM. Hermetia illucens larvae meal can be a suitable alternative protein source for laying hens even if the complete replacement of soybean meal needs further investigation to avoid the negative effects on feed intake.

Expression and Localization of Aquaporin-1 Along the Intestine of Colostrum Suckling Buffalo Calves

Aquaporin‐1 (AQP1), a six‐transmembrane domain protein, belongs to a highly conserved group of proteins called aquaporins known to regulate permeability across cell membranes. Although the role of AQP1 has been extensively studied, its specific activity along the gastrointestinal tract in animals during early postnatal development is poorly known. This study investigates the expression of AQP1 mRNA and protein in the small and large intestine of water buffalo calves after colostrum ingestion using reverse transcription–polymerase chain reaction (RT‐PCR), Western blotting, and cellular localization of AQP1 by immunohistochemistry. Our results revealed AQP1 immunoreactivity and the presence of the corresponding mRNA in all the examined tracts of the intestine but with a different cellular localization. Western blotting confirmed the presence of AQP1, with a more intense band in colostrum‐suckling animals. These findings offer insights into AQP1 expression in the small and large intestine, suggesting its involvement in osmoregulation in gastrointestinal physiology particularly during the first week after birth in relation to specific maturation of intestinal structures.

Expression and Localization of Aquaporin 4 and Aquaporin 5 along the Large Intestine of Colostrum‐Suckling Buffalo Calves

Aquaporins (AQPs) are membrane channel proteins that play a role in regulating water permeability in many tissues. To date, seven isoforms of AQPs have been reported in the gastrointestinal tract in different mammalian species. In contrast, both tissue distribution and expression of AQPs are unknown in the buffalo. The purpose of this study was to investigate the expression of both AQP4 and AQP5 mRNAs and their relative proteins in the large intestinal tracts of buffalo calves after colostrum suckling using reverse transcriptase polymerase chain reaction (RT‐PCR), Western blotting and immunohistochemistry. Our results revealed a diversified tissue AQP4 and AQP5 immunolocalization accompanied by their highest expression in the tissues of colostrum‐suckling buffalo calves confirmed by Western blotting. In particular, AQP4 was distributed along the endothelium and enterocytes while AQP5 in the endocrine cells. These findings provide direct evidence for AQP4 and AQP5 expression in the large intestine, suggesting that different AQPs collaborate functionally and distinctively in water handling during intestinal development, especially during the first period after delivery.

Haemostatic disorders in dogs naturally infected by Leishmania infantum.

1M. Corona1, P. Ciaramella1*, A. Pelagalli2, L. Cortese1, M.E. Pero2, D. Santoro1 and P. Lombardi2 1Department of Veterinary Clinical Science – Section of Internal Medicine; 2Department of Biological Structure, Function and T echnologies – University of Naples Federico II, via Delpino, 1-80137 Naples, Italy *Correspondence: Dipartimento di Scienze Cliniche Veterinarie – Sezione di Clinica Medica – Facoltà di Medicina Veterinaria, via Delpino, 1-80137 Napoli, Italy E-mail: paociara@unina.it

Urocortin-like immunoreactivity in the primary lymphoid organs of the duck (Anas platyrhynchos)

Urocortin (UCN) is a 40 aminoacid peptide which belongs to corticotropin-releasing factor (CRF) family. This family of peptides stimulates the secretion of proopiomelanocortin (POMC)-derived peptides, adrenocorticotropic hormone (ACTH), β-endorphin and melanocyte-stimulating hormone (MSH) in the pituitary gland. In the present study, using Western blotting and immunohistochemistry, the distribution of UCN in the primary lymphoid organs of the duck was investigated at different ages. In the cloacal burse and thymus, Western blot demonstrated the presence of a peptide having a molecular weight compatible with that of the mammalian UCN. In the cloacal burse, immunoreactivity was located in the medullary epithelial cells and in the follicular associated and corticomedullary epithelium. In the thymus, immunoreactivity was located in single epithelial cells. Double labelling immunofluorescence studies showed that UCN immunoreactivity completely colocalised with cytokeratin immunoreactivity in both the thymus and cloacal burse. Statistically significant differences in the percentage of UCN immunoreactivity were observed between different age periods in the cloacal burse. The results suggest that, in birds, urocortin has an important role in regulating the function of the immune system.

Gammaglutamyltransferase activity in buffalo mammary tissue during lactation

In the present study, the role of gammaglutamyltransferase (GGT) during lactation has been investigated in the water buffalo. GGT activity has been evaluated in the mammary tissue at 4 and 6 months after calving and during the non- lactating period. The highest GGT activity levels were found at day 120 (32·57 ^ 7·41U per g) of lactation and were statistically higher than those at 180 (10·76 ^ 3·6U per g) or during the non-lactating period (9·86 ^ 7·94U per g). Histochemistry confirmed these findings and revealed that GGT reactivity was distributed throughout the cytoplasm of alveolar epithelial cells. Such results showed that the GGT production is high during lactation thus supporting the hypothesis that this enzyme plays a role in determining milk production in water buffalo by supporting milk protein synthesis.

Effect of colostrum and milk on small intestine expression of AQP4 and AQP5 in newborn buffalo calves

Functional studies indicate differences in newborn gastrointestinal morphology and physiology after a meal. Both water and solutes transfer across the intestinal epithelial membrane appear to occur via aquaporins (AQPs). Given that the physiological roles of AQP4 and AQP5 in the developing intestine have not been fully established, the objective of this investigation was to determine their distribution, expression and respective mRNA in the small intestine of colostrums-suckling buffalo calves by using immunohistochemistry, Western blot, and reverse transcriptase-PCR analysis. Results showed different tissue distribution between AQP4 and AQP5 with the presence of the former along the enteric neurons and the latter in the endocrine cells. Moreover, their expression levels were high in the ileum of colostrum-suckling buffalo calves. The data present a link between feeding, intestinal development and water homeostasis, suggesting the involvement of these channel proteins in intestinal permeability and fluid secretion/absorption during this stage of development after birth.

Laying performance, blood profiles, nutrient digestibility and inner organs traits of hens fed an insect meal from Hermetia illucens larvae

Given probable the increment in the nutritional needs of both humans and animals, animal production will have increased dramatically by 2050. Insect meals could be an alternative protein source for livestock, and they would also be able to reduce the environmental problems related to intensive animal production system. The aim of this study was to evaluate productive performance, blood analysis, nutrient digestibility, and changes in the internal organs of laying hens fed Hermetia illucens larvae meal (HI) at two different levels in substitution (25 or 50%) of soybean meal (SBM). A total of 162 Hy-line Brown hens (sixteen weeks old) were equally divided into three experimental groups and fed isoprotein and isoenergetic diets. Egg weight, feed intake, and feed conversion rate were not affected by the soybean meal substitution at both inclusion levels of insect meal. Egg mass was positively affected by the insect meal diets, as was the lay percentage, although only at the lowest inclusion level. Dry matter, organic matter, and crude protein digestibility coefficients were lower for the HI50 diet, probably due to the negative effect of chitin. A reduction in serum cholesterol and triglycerides was observed in both insect-meal fed groups, while serum globulin level increased only at the highest level of insect meal inclusion, and, consequently, the albumin to globulin ratio decreased. Overall, a protein replacement of 25% with an insect meal from Hermetia illucens larvae in the diet of laying hens seems to be more suitable and closer to the optimal level.

51 CASPASE-3 INHIBITOR Z-VAD-FMK ENHANCES CRYOTOLERANCE OF IN VITRO-PRODUCED BOVINE PRE-IMPLANTATION EMBRYOS

In vitro-produced (IVP) bovine embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. Cryopreservation induces cell degeneration through the apoptotic pathway in bovine oocytes and embryos (Men et al. 2003 Cryobiology 47, 73-81). Apoptosis can be prevented by inhibition of caspase activity, leading to improved cryosurvival in mammalian cells (Stroh et al. 2002 FASEB J. 16, 1651-3). Interestingly, cryotolerance of porcine embryos was improved by inhibiting apoptosis using a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during vitrification and subsequent culture (Men et al. 2006 Theriogenology 66, 2008-16). Aim of this work was to evaluate whether cryotolerance of bovine IVP embryos may be improved by using Z-VAD-FMK during cryopreservation and post-warming in vitro culture. Abattoir-derived bovine oocytes (n=753, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-55). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium at 39°C with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and blastocysts (except the hatched blastocysts) were randomly divided in 2 groups: vitrification and post-warming culture in presence (n=60) or absence (n=54) of 20µM Z-VAD-FMK. Vitrification was carried out by Cryotop in 16.5% ethylene glycol, 16.5% DMSO, and 0.5M sucrose (Rubessa et al. 2011 Theriogenology 76, 1347-55). Blastocysts were warmed in decreasing sucrose solutions (0.25M for 1min and 0.15M for 5min) and cultured for 2 days. Resistance to cryopreservation was evaluated by assessing the survival rate, based on morphological criteria and hatching rate after 48h culture. Furthermore, TUNEL staining was used to evaluate the total cell (TC) number and the apoptotic rate of vitrified blastocysts after 48-h post-warming culture. Differences between groups in survival and hatching rates after 48-h post-warming culture were analysed by Chi-squared test, whereas differences in TC number and in number and percentage of apoptotic cells were analysed by Students t-test. Inhibition of caspase activity induced by Z-VAD-FMK increased embryo cryotolerance, as indicated by higher survival (92.6v. 55.0%; P<0.01) and hatching rates (40.7v. 23.3%; P<0.05) after 48h of post-warming culture. Furthermore, Z-VAD-FMK decreased both the average number (7.1±0.6v. 4.2±0.3; P<0.01) and the percentage (6.3±0.6v. 3.0±0.2; P<0.01) of apoptotic cells in blastocysts. No differences were recorded in TC number between groups (on average, 128.90±1.6). These results suggest that addition of 20µM Z-VAD-FMK during vitrification/warming and post-warming culture significantly inhibits apoptosis (DNA fragmentation) and improves the cryotolerance of IVP bovine embryos.

Influence of γ-glutamyltransferase and alkaline phosphatase activity on in vitro fertilisation of bovine frozen/thawed semen

Abstract The aim of this work was to evaluate whether the residual amount of γ-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) in bovine sperm after freezing/thawing is correlated with fertility parameters, including blastocyst rates after in vitro fertilisation (IVF). The enzyme activities were determined in both spermatozoa and supernatant after centrifugation. While ALP was only correlated with sperm viability, GGT activity was correlated with sperm motility (rs = .4; p < .05) both in sperm and supernatant. Interestingly, GGT activity was also correlated with cleavage (rs = .5; p < .05 and .8; p < .01, for sperm and supernatant respectively) and blastocyst (rs = .6 and .9, for sperm and supernatant respectively; p < .01) rates obtained after IVF. These results suggest that GGT could play an important role in the protection of sperm against oxidative stress and could be considered a reliable marker to assess frozen/thawed sperm quality in bovine.